Certain endogenous steroids and their synthetic analogues (neuroactive steroids) produce profound and rapid effects on the central nervous system ranging from general anesthesia to seizures. While these effects are thought to result from steroid interactions with specific binding sites on the GABA-A receptor, molecular biological studies have failed to identify candidate regions or residues that might contribute to a binding site. The overall goal of this project is to identify and characterize the binding sites with which neuroactive steroids interact to produce their inhibitory (anesthetic) effects. Two novel neurosteroid analogue photoaffinity labeling reagents, CW12 (a Sa-reduced steroid) and CW14 (a 5p-reduced steroid) have been developed to achieve this goal. Both CW12 and CW14 are potent and efficacious modulators of GABA-A receptor function. When these reagents are exposed to UV light at 350 nM, they form a reactive group (a carbene) that allows them to covalently attach to (photolabel) their binding site. Assays of GABA-A receptor function in brain membranes provide strong evidence that CW12 and CW14 efficiently and selectively photolabel the neuroactive steroid binding site(s) responsible for modulation of GABA-A receptor function. This project has two specific aims. The first is to identify and characterize the GABA-A receptor-associated proteins that are photolabeled by CW12 and/or CW14 in rat brain. The second aim is to determine the GABA-A receptor subunit specificity of neuroactive steroid binding and action by photolabeling HEK cells transfected with various combinations of epitope-tagged GABA-A receptors and correlating the functional effects of photolabeling with the specific subunits that are photolabeled. In both aims, photolabeled proteins will be isolated using immunoprecipitation and two-dimensional electrophoresis and then identified and sequenced using tandem mass spectrometry. The information gained from this project will provide the background knowledge and tools to: (1) determine how endogenous neurosteroids modulate CNS function in health and disease and; (2) develop new pharmaceutical agents including potent steroidal anesthetics with minimal side effects, novel anticonvulsants and anxiolytics and neuroactive steroid antagonists. [unreadable] [unreadable]